2.2. Generation of Spheroids, Organoids, and hPEK-coated Microcarrier Beads

3D spherical cultures consisting of one cell type (hMSCs) or an unorganized mixture of two cell types (as discussed in the Supporting Information section) will be referred to as spheroids. 3D cultures consisting of multiple cell types (hMSC+hPEK) in an organized, layered architecture that depicts the architecture of the palate will be referred to as organoids.

Spheroids were generated in microwells of agarose dishes. Dishes were made by pouring molten 2% solution of UltraPure™ Agarose (Invitrogen, Carlsbad, CA) into a MicroTissues® 3D Petri Dish ® (Sigma-Aldrich, St. Louis, MO) containing either 5×7 (#Z764051, Sigma-Aldrich) or 16×16 (#Z764000) grid microwells and cooling for 15 min. Agarose dishes were transferred to a 24 or 12 well culture plate in 500 µl hMSC-G and equilibrated at 37° C and 5% CO₂ for 30 min.

hMSCs at passage 3 – 10 were counted and either left unstained or stained with CellTracker™ (CT) orange CMTMR (Invitrogen) at 10 µM for 25 min, pelleted, rinsed in sterile Dulbecco’s phosphate buffered saline (DPBS) and resuspended in hMSC-G with 0.2% Penicillin/Streptomycin/Amphotericin B (P/S/A; ATCC). Agarose dishes were drained of media and 75 µl hMSCs in hMSC-G were seeded into dishes at various cell densities. Cells were allowed to settle into microwells for ~15 min, medium was restored to each well to cover the agarose dish, and the plate was placed in incubator overnight. hMSC spheroids formed overnight and the next day, day 1 (d1), medium was changed to various ratios of O medium and keratinocyte differentiation (K) medium (CellnTec) for experimentation, or to StemPro® Osteogenic Differentiation (O) medium (Gibco, Gaithersburg, MD) with 0.5% P/S/A and 10µg/ml Kanamycin, and cultured up to 20 days. For the final spheroid model, hMSCs were formed in hMSC-G medium overnight, and cultured in O medium from d1-d13.

To generate heterotypic spheroids and organoids, hMSC spheroids were coated with 50% Maxgel™ (MG) extracellular matrix (Sigma-Aldrich) by immersing spheroids in cold 1:1 MG:hMSC-G medium mixture for 2 h, rinsing with DPBS, and transferring spheroids by pipette to a fresh agarose dish in hMSC-G medium. hPEKs were harvested from passage 3–10, suspended in either hPEK-G, K, or serum-free epithelial/stromal co-culture medium (CC; #CnT-PR-CC, CellnTec), and either left unstained or stained with CellTracker™ green CMFDA (Invitrogen). Dishes of hMSC spheroids were drained of media and 75µl of hPEK cell suspension at various densities was seeded onto hMSC spheroids in the agarose dish on d2, d8, or d13 of hMSC culture. After settling of hPEKs, appropriate media was restored to cover dishes and spheroids with media. Spheroids and cells were incubated at 37° C and 5% CO² in a humid environment on a rotator at a gentle rotation (~60 rpm) for 6 −24 h (d0). Heterotypic spheroids were completely formed as organoids, with hPEKs attached to the outer surface of the hMSC core, by the next day (d1). See Supporting Information for spheroids generated from co-suspension of hMSCs and hPEKs.

CT green-stained hPEKs suspended in hPEK-G were seeded onto Corning® enhanced-attachment microcarrier polystyrene beads (Corning #3779; Sigma-Aldrich) at 320,000 cells to 350 beads in a microcentrifuge tube, transferred to a well of a 96-well culture plate, rotated (swirled) at ~150 rpm for 4–5 h, and 100 µl hPEK-G was added to the well. hPEK-seeded beads were cultured in conditions described for spheroids to produce hPEK-coated microcarrier beads.